Cosmetic treatment method for fighting against skin ageing effects

ABSTRACT

The present invention relates to a method of cosmetic treatment for combating the effects of skin ageing and to novel cosmetic compositions which are particularly suitable for carrying it out. According to the invention, at least one agent for promoting the adhesion of the keratinocytes of the epidermal baseal layer to the dermo-epidermal junction, especially to the collagen IV of said junction, such as, in particular, a divalent metal salt or complex, preferably magnesium aspartate or magnesium chloride, is used, optionally in association with a stimulant of collagen IV synthesis and/or a stimulant of collagen VII synthesis. The application is for the preparation of cosmetic compositions with anti-wrinkle activity.

This application is a 371 of International Application No.PCT/FR99/01261, filed May 28, 1999, which claims priority tocontinuation-in-part U.S. application Ser. No. 09/297,679, now U.S. Pat.No. 6,193,975 , filed May 6, 1999, French Application No. 98/06822,filed May 29, 1998, and French Application No. 96/13585, filed Nov. 7,1996.

The present invention relates to a method of cosmetic treatment forcombating the effects of skin ageing and to novel cosmetic compositionswhich are particularly suitable for carrying it out.

The dermo-epidermal junction (DEJ) is known to be a complex structureassuring the cohesion and exchanges between the dermis and epidermiswhich are essential for the skin to function properly.

It has been discovered that it is possible to slow down or treat skinageing, and in particular to reduce the depth of wrinkles, and/or slowdown their appearance, and/or restore the tonicity and elasticity of theskin, and/or slow down the decrease in tonicity and elasticity of theskin, by means of a method of cosmetic treatment corresponding to anovel concept which consists in using a cosmetically acceptable agent topromote the adhesion of the keratinocytes of the epidermal basal layerto the dermo-epidermal junction, especially to the type IV collagen,also called collagen IV, which is a major constituent of saiddermo-epidermal junction. It is this discovery which constitutes thebasis of the present invention.

Thus, according to its most general feature, the present patentapplication aims to cover a method of cosmetic treatment for slowingdown or treating skin ageing, and in particular for reducing the depthof wrinkles, and/or slowing down their appearance, and/or restoring thetonicity and elasticity of the skin, and/or slowing down the decrease intonicity and elasticity of the skin, characterized in that an amount ofat least one agent for promoting the adhesion of the keratinocytes ofthe epidermal basal layer to the dermo-epidermal junction, especially tothe collagen IV of said junction, is applied to the skin.

It has furthermore been shown that particularly remarkable results areobtained within the framework of the present invention if theabove-mentioned adhesion promoter is applied in association with aneffective amount of at least one stimulant of collagen IV synthesisand/or with an effective amount of at least one stimulant of collagenVII synthesis.

The expression “stimulant of collagen IV or collagen VII synthesis” isunderstood within the framework of the present description as meaningany agent which is capable of producing or maintaining a high level ofcollagen IV in the dermo-epidermal junction, either by increasing thebiosynthesis or by inhibiting the enzymes which degrade the constituentproteins of this product.

In one advantageous embodiment of the invention, the above-mentionedadhesion promoter is a divalent metal salt or complex, particularly amagnesium or zinc salt or complex, or a mixture of divalent metal saltsor complexes.

The divalent metal salt or complex is preferably a divalent metalchloride or a divalent metal salt or complex with a cosmeticallyacceptable organic acid such as an amino acid, for example asparticacid, asparagine, proline, glutamic acid, methionine, leucine, histidineor lysine, or a C₂-C₁₂ aliphatic alpha-hydroxy acid, particularly citricacid, glycolic acid, gluconic acid, malic acid, lactic acid or2-hydroxybutyric acid.

In one currently preferred embodiment of the invention, said divalentmetal salt or complex is magnesium aspartate or magnesium chloride.

According to one particular characteristic of the method of the presentinvention, the above-mentioned adhesion promoter is applied in the formof a composition in which it is present in an amount of between 0.0001and 5% by weight, preferably of between 0.001 and 1% by weight, based onthe total weight of the composition.

Any stimulant of collagen IV synthesis can be used within the frameworkof the method according to the present invention.

In one currently preferred embodiment, the stimulant of collagen IVsynthesis is selected from soya saponins and soya sapogenols, preferablyof type A and type B, and plant extracts rich in such compounds,preferably extracts of soya (Glycine max) or alfalfa (Medicago sativa).

In another preferred embodiment, the stimulant of collagen IV synthesisis a whole range of saponins from roots of Medicago sativa.

Likewise, any stimulant of collagen VII synthesis can be used within theframework of the present invention.

In one currently preferred embodiment, the stimulant of collagen VIIsynthesis is an extract of Potentilla erecta.

In another preferred embodiment, the stimulant of collagen VII synthesisis an extract of Bertholletia, particularly Bertholletia excelsa.

According to a second feature, the present patent application aims tocover novel cosmetic compositions which are particularly suitable forcarrying out the method described above.

These compositions are essentially characterized in that they contain aneffective amount of at least one agent for promoting the adhesion of thekeratinocytes of the epidermal basal layer to the dermo-epidermaljunction, especially to the collagen IV of said junction, said agentbeing selected from magnesium or zinc salts or complexes, in associationwith an effective amount of at least one stimulant of collagen IVsynthesis and/or an effective amount of at least one stimulant ofcollagen VII synthesis.

In these compositions, the various adhesion promoters and stimulants ofcollagen IV synthesis or collagen VII synthesis are as described abovewithin the framework of the general description of the method accordingto the invention.

The compositions of the invention may also advantageously comprise atleast one substance for promoting the synthesis of the constituents ofthe extracellular matrix of the skin.

Furthermore, the compositions according to the invention can alsocontain at least one substance selected from the group consisting ofvitamins, particularly the vitamins of group A (retinol) and group C andderivatives thereof such as the esters, especially the palmitates andpropionates, tocopherols, xanthines, particularly caffeine ortheophylline, retinoids, particularly vitamin A acid, extracts ofCentella asiatica, asiatic and madecassic acids and glycosylatedderivatives thereof such as asiaticoside or madecassoside, extracts ofSiegesbeckia orientalis, extracts of Commiphora mukul and extracts ofEriobotrya japonica, cosmetically acceptable silicon derivatives such aspolysiloxanes, silanols and silicones, C₃-C₁₂ aliphatic alpha-ketoacids, particularly pyruvic acid, C₂-C₁₂ aliphatic alpha-hydroxy acids,particularly citric acid, glycolic acid, malic acid and lactic acid,amino acids, particularly arginine, citrulline and threonine, ceramides,glycoceramides, sphingosine derivatives, particularly type II and IIIceramides, phospholipids, forskolin and derivatives thereof, extracts ofColeus, extracts of Tephrosia, elastase inhibitors, particularly ellagicacid and soya peptides, collagenase inhibitors, particularly plantpeptides and extracts such as extracts of roots of Coptidis and extractsof roots of Scutellaria baicalensis Georgi, flavonoids such as wogonin,baicalin and baicalein, aqueous-ethanolic extracts of leaves of Ginkgobiloba, Mosla chinensis, Salvia officinalis and Cinnamomum cassia,catechuic extracts of Camellia sinensis and aqueous extracts of beanshells of Theobroma cacao, anti-inflammatories, particularlyphospholipase A2 inhibitors, soothing agents, particularly extracts ofliquorice, glycyrrhetinic acid and ammonium glycyrrhizinate, hydratingagents, particularly polyols, propylene glycol, butylene glycol,glycerol and hyaluronic acid, agents for combating stretch marks,particularly extracts of horse chestnut and escin, agents for protectingor improving the microcirculation, particularly bioflavonoids fromGinkgo biloba, isodon, extracts of Ami visnaga, visnadine andruscogenin, free radical inhibitors, particularly polyphenols such asPCO (procyanidolic oligomers) and derivatives thereof and plantextracts, particularly extracts of Curcuma longa, antiseborrhea agents,such as a 5-alpha-reductase inhibitor, particularly an extract of Pygeumafricanum, and stimulants of the microcirculation of the blood, such ascepharanthine and methyl nicotinate.

The compositions according to the invention can advantageously containsubstances for protecting the skin from the harmful effects of the sun,such as solar filters, individually or in combination, especially UV Afilters and UV B filters, particularly titanium oxides and zinc oxides,oxybenzone, Parsol MCX, Parsol 1789 and filters of vegetable origin,substances for limiting the damage caused to the DNA, particularly thosefor limiting the formation of thymine dimers, such as ascorbic acid andderivatives thereof and/or Photonyl®, and substances for contributing tothe elimination of liver spots, such as inhibitors of melanin ortyrosinase synthesis.

Other objects, characteristics and advantages of the invention willbecome clearly apparent to those skilled in the art from the followingexplanatory description referring to several Examples relating to testsperformed, and Examples of cosmetic formulations, which are given simplyby way of illustration and cannot therefore in any way limit the scopeof the invention.

ALL THE PERCENTAGES ARE GIVEN BY WEIGHT IN THE EXAMPLES, UNLESSINDICATED OTHERWISE. EXAMPLE 1 Test for the Adhesion of Normal HumanKeratinocytes to Type IV Collagen with the Aid of an Agent According tothe Invention for Promoting the Adhesion of the Keratinocytes to theDermo-epidermal Junction, Consisting of Magnesium Chloride or Aspartatein this Test

The object of the present test is to demonstrate the efficacy of anagent according to the invention for promoting the adhesion of thekeratinocytes to the dermo-epidermal junction, said agent preferablyconsisting of magnesium chloride or aspartate.

Within this framework, said test is carried out in the following manner:

1. Coating of the Adhesion Surfaces with type IV Collagen

Wells of microplates (Falcon) are covered with 6 μg/cm² of sterile typeIV human collagen (from Sigma).

Each well is then incubated overnight at +4° C. with a 4 mg/ml solutionof bovine albumin, BSA, from Sigma.

The wells are then rinsed twice with a phosphate buffer, PBS (phosphatebuffered saline), from Gibco.

2. Preparation of the Cultures of Normal Human Keratinocytes

The epidermal cells are obtained from healthy surgical skin originatingfrom the mammary region of a 53-year-old female caucasian donor.

The skin fragments are incubated in 0.25% w/v trypsin for 18 hours at+4° C. to separate the dermis and epidermis and to obtain, by agitation,a suspension of epidermal cells. The trypsin is neutralized with fetalcalf serum, FCS, from Gibco.

The cells are inoculated into flasks defining a surface area of 75 cm²and are cultured in keratinocyte proliferation medium, K-SFM, fromGibco, to the point of confluence, when they are subcultured.

The cells used for the experiments for adhesion to the collagensubstrate were not subcultured beyond the first subculture (called P0 orP1).

3. Treatment of the Keratinocytes with the Product of the Invention,Consisting of either Magnesium Chloride or Magnesium Aspartate

Trypsin (trypsin containing 0.1% -0.02% w/v of EDTA, from Gibco) isadded to the keratinocyte cultures, and the cell suspension is placed inE 199 medium from Gibco, complemented with 2 mM L-glutamine and 4 mg/mlof BSA and containing, according to the invention, 0.25, 0.5 or 1 mMmagnesium chloride or 0.25 mM magnesium aspartate.

The keratinocytes are then incubated for 30 minutes at +4° C. before thestep for adhesion to the substrate coated with collagen IV is carriedout.

4. Measurement of the Adhesion of the Keratinocytes to the Type IVCollagen

The keratinocytes are inoculated into each well at a density of 93,000cells/cm² in E 199 medium from Gibco, containing 2 mM L-glutamine fromGibco and 4 mg/ml of BSA (bovine serum albumin). After incubation forone hour at +4° C., the wells are rinsed with PBS, the adhering cellsare then lyzed with 0.1 N sodium hydroxide solution and the cellularproteins are then quantified by means of the colorimetric method andbicinchoninic acid (BCA from Sigma).

A calibration is performed in parallel with BSA solubilized in 0.1 Nsodium hydroxide solution, enabling the optical density (OD) values tobe converted to micrograms (μg) of proteins per well.

5. Statistical Analysis

The adhesion A is expressed in micrograms of cellular proteins perculture well and the values shown in Table I correspond to a mean valueobtained from 6 wells per product concentration, the products being theuntreated cells, the cells treated with a magnesium chloride oraspartate concentration of 0.25 mM, the cells treated with a magnesiumchloride concentration of 0.5 mM and the cells treated with a magnesiumchloride concentration of 1 mM.

These adhesion values between treated and untreated cells were comparedby the Student t-test at the p=0.05 threshold in order to assess theirlevel of significance.

The results obtained from the experiment on the keratinocytes of a53-year-old donor are listed in Table I below:

TABLE I A Standard deviation Student t-test Control 3.52 0.9 Magnesiumchloride 4.32 0.7 Not significant (0.25 mM) (p = 0.12) Magnesiumchloride 4.54 1.1 Not significant (0.5 mM) (p = 0.1) Magnesium chloride4.57 0.75 Significant (1 mM) (p = 0.05) Magnesium aspartate 5.39 0.8Significant (0.25 mM) (p = 0.004) A = adhesion in μg of protein per well(mean)

The results listed in Table I above show that, compared with the controlcultures, there is an increase in the adhesion of the keratinocytes tothe type IV collagen in the presence of magnesium chloride as from the0.25 mM concentration, but this is only statistically significant asfrom 1 mM.

The percentage increase in adhesion obtained with magnesium chloride atthe 1 mM concentration is +31.

As far as magnesium aspartate is concerned, this also promotes theadhesion of the keratinocytes, but more strongly than magnesiumchloride. In fact, the increase in adhesion is highly significant asfrom the 0.25 mM concentration.

The percentage increase in adhesion obtained with magnesium aspartate atthe 0.25 mM concentration is +54.

Under these conditions, it is thus seen that these magnesium salts, andmore especially magnesium aspartate, are of particular value becausethey produce highly significant results at low doses, enabling them tobe used at low concentrations and hence with a good degree of safety.

EXAMPLE 2 OF THE INVENTION

1. Composition of an anti-wrinkle cream Magnesium L-aspartate 0.3 g Dryextract of Potentilla erecta 0.01 g Hyaluronic acid (sodium salt) 0.06 gGlycerol 5.15 g Total dry extract of Centella asiatica 0.1 g Vitamin Apalmitate solution (1 million IU/g) 0.1 g Vitamin E acetate 0.5 g Dryextract of Perilla 0.5 g O/W emulsion excipient plus perfume andpreservatives qsp 100 g

2. Testing of this Cosmetic Composition for Evaluation of itsAnti-wrinkle Efficacy

A—Principle

To evaluate the anti-wrinkle efficacy of this cosmetic product on“crow's feet”, negative replicas of skin are made at time 0 and thenafter 28 days of twice daily application of the above composition in theform of a cream.

These replicas, illuminated by a glancing light casting shadows behindeach wrinkle, are analyzed with the aid of a commercially availableimage-analyzing software, called “Quantirides”, developed by MONADERM(Monaco).

B—Equipment

B.1—For taking Impressions

Adhesive rings from 3M, of internal diameter 24 mm and external diameter40 mm, are used.

The product Silflo® from FLEXICO UK, based on a silicone polymercombined with a catalyst, is used to take impressions.

B.2—For analyzing the Impressions

The following are used:

a COHU 4910-RS 170 and CCIR Monochrome CCD camera, which is ahigh-definition and very low-noise camera equipped with a fixed-focuslens and an 18×108 mm F 2.5 manual zoom lens;

a real-time high-resolution image acquisition card;

a Monaspot glancing illumination lamp with an angle of incidence of 35°;

a Kaiser RS 1 tripod, a 450×500 mm anti-reflection matt black plate anda 1000 mm height-adjustable column graduated in centimeters, equippedwith an RA1 projection arm;

a special support for positioning and orientating the replicas;

the above-mentioned Quantirides software; and

a microcomputer and a printer.

C—Protocol

1. Volunteers

30 subjects aged between 34 and 59 years, comprising 29 women and 1 man,were selected.

2. Test Product

The test product is the composition in the form of a cream describedabove in section 1.

3. Application

The cosmetic composition is applied twice a day, in the morning andevening, to the whole of one temporal zone of the face (crow's foot) for28 days. The amount applied can be estimated at about 1.5 to 2 mg per cm2, depending on what the subject is accustomed to. The other,“untreated” temporal zone serves as the control.

For three days preceding the start of the test, and throughout theentire test, no other cosmetic product is used on the treated zone orthe control zone.

4. Experimental Conditions

Temperature: from 20 to 22° C.

Relative Humidity: from 40 to 50%

An impression of the control and treated zones is made at time 0 andafter 28 days of treatment. An adhesive ring is positioned over thestudy zone. A thin layer of Silflo®, mixed with a few drops of catalyst(3 drops per 3 g of Silflo®) immediately before use, is applied to theinside of the zone delimited by the ring. The paste must be spreadcarefully to avoid the formation of air bubbles. After polymerization ofthe paste, a drying time of 4 minutes 30 seconds, the ring is detachedfrom the skin, bringing the replica with it. At the end of the study,these impressions are analyzed with the aid of the Quantirides software.

D. Parameters Studied

For each subject and for each side of the face, on D0 (the day beforethe first application) and D28 (day 28 of application) processing of theimpressions by the image analyzer made it possible to calculate thefollowing parameters representing the state of wrinkling of the skin:

a) the total surface area of the wrinkles in mm²;

b) the number of wrinkles;

c) the total length of the wrinkles in mm;

d) the mean length in mm; and

e) the mean depth in μm.

E. Processing of the Results: Change in the Treated Side and the ControlSide

1. Calculation

Mean Variation of the Parameters

The following is calculated for each site and each parameter:${\text{Variation} \cdot (\%)} = {\left( \frac{{m(t)} - {m(0)}}{m(0)} \right) \times 100}$

where

m(t)=mean value of the parameter studied at time t

m(0)=mean value of the parameter studied at time 0

2. Statistical Significance

Wilcoxon Test

The non-parametric Wilcoxon test is used, which makes it possible totake account of the small number of subjects and is applicable to the invivo study of biological parameters in humans.

A comparison of the paired series is made as follows: the difference isevaluated for each pair and the differences are then placed inincreasing order of absolute value; it is also indicated for each onewhether it is positive or negative, zero differences being eliminated.

The quantities to be considered are as follows:

M=sum of the ranks of negative difference

P=sum of the ranks of positive difference

T=the smaller of the two totals, M or P

The significance limit accepted for n<10 persons is below 10%.

The significance limit accepted for n≧10 persons is below 5%.

The Wilcoxon test was applied to the difference (m(t)−m(0)) at thedifferent times at the two sites in order to compare the change in thetreated site with the change at the control site.

A Wilcoxon test was applied to the raw values at time 0 (m(0)) in orderto compare the change in each site with time.

3. Results

The results obtained from the experiment are listed in Table II below.

The control and treated sites are comparable at time 0.

The values shown in column 4 under the heading “total variation”correspond to the difference between the variation in the treatedsubjects and the variation in the control subjects.

TABLE II Variation Variation in in control treated Total subjectssubjects variation Statistics % % % (Wilcoxon) Total surface area 6.6−19.7 −26.3 Significant of wrinkles (mm²) (p = 0.015) Number of 16.5−14.3 −30.8 Significant wrinkles (p = 0.004) Total length (mm) 12.9−16.7 −29.6 Significant (p = 0.0014) Mean length (mm) −2.6 −1.7 0.9 Notsignificant Mean depth (μm) 0.3 −2.0 −2.3 Not significant

E. Conclusion

After twice daily application of the composition according to theinvention for 28 days, the change in the treated crow's foot is comparedwith the change in the control crow's foot to reveal a significantdecrease in the large and small wrinkles and a slowing-down of theirformation: the total surface area of the wrinkles drops by 26%, theirnumber by 31% and their total length by 30%.

EXAMPLE 3 OF THE INVENTION

W/O Anti-Wrinkle Night Cream

Magnesium aspartate 0.3 g Dry extract of Potentilla erecta 1 g Glycerol5 g Propylene glycol 2 g Ceramide III 0.04 g UV filters 9 gMethylsilanol mannuronate 0.05 g Dry extract of Perilla frutescens 1 gDry extract of Centella asiatica 0.5 g Soya peptide 1 g Retinolpalmitate 0.2 g W/O emulsion excipient qsp 100 g

EXAMPLE 4 OF THE INVENTION

Firming Cream for Slowing Down and Combating the Appearance of Wrinkles

Magnesium aspartate 0.2 g Glycerol 5 g Propylene glycol 2 g Ceramide II0.04 g Parsol MCX 5 g Oxybenzone 3 g Methylsilanol mannuronate 0.05 gMadecassoside 0.5 g Retinol 4000 IU Saponins from Medicago sativa 0.02 gRetinol palmitate 0.04 g O/W emulsion excipient qsp 100 g

EXAMPLE 5 OF THE INVENTION

Anti-wrinkle Tightening Gel

Zinc gluconate 0.3 g Dry extract of Bertholletia excelsa 0.3 g Soyasaponin 0.05 g Retinol palmitate 0.06 g Alpha-tocopherol acetate 0.1 gLactic acid 1.5 g Glycolic acid 0.2 g Ethanol 5 g Gel excipient qsp 100g

What is claimed is:
 1. A method of cosmetic care for promoting theadhesion of the keratinocytes of the epidermal basal layer to thedermo-epidermal junction, comprising the steps of determination of theparts of the skin in need thereof and application to said parts of acosmetic composition containing an active agent selected from the groupconsisting of magnesium salts, magnesium complexes, and mixturesthereof, in an efficient amount for promoting said adhesion.
 2. Themethod according to claim 1, wherein said active agent is a magnesiumsalt.
 3. The method according to claim 2, wherein said magnesium salt ismagnesium chloride.
 4. The method according to claim 1, wherein saidactive agent is a complex of magnesium with a cosmetically acceptableorganic acid.
 5. The method according to claim 4, wherein said complexis a complex of magnesium with an amino acid.
 6. The method according toclaim 5, wherein said complex of magnesium is a complex with an aminoacid selected from the group consisting of aspartic acid, asparagineacid, proline, glutamic acid, methionine, leucine, histidine, andlysine.
 7. The method according to claim 6, wherein said magnesiumcomplex is magnesium aspartate.
 8. The method according to claim 4,wherein said complex is a magnesium complex with a C₂-C₁₂ aliphaticalpha-hydroxy acid.
 9. The method according to claim 8, wherein saidC₂-C₁₂ aliphatic alpha-hydroxy acid is selected from the groupconsisting of citric acid, glycolic acid, gluconic acid, malic acid,lactic acid, and 2-hydroxybutyric acid.
 10. The method according toclaim 1, wherein said composition comprises between 0.0001 and 5% byweight of said active agent, based on the total weight of thecomposition.
 11. The method according to claim wherein said compositioncomprises between 0.01 and 1% by weight of said active agent, based onthe total weight of the composition.
 12. The method according to claim1, wherein said composition further comprises an effective amount of atleast one agent stimulating the synthesis of collagen IV.
 13. The methodaccording to claim 12, wherein said agent stimulating the synthesis ofcollagen IV is selected from the group consisting of soya saponins, soyasapogenols, and mixtures thereof.
 14. The method according to claim 12,wherein said agent stimulating the synthesis of collagen IV is anextract from roots of Medicago sativa.
 15. The method according to claim1, wherein said composition further comprises an active agentstimulating the synthesis of collagen VII.
 16. The method according toclaim 15, wherein said active agent stimulating the synthesis ofcollagen VII is an extract of Potentilla erecta.
 17. The methodaccording to claim 15, wherein said active agent for stimulating thesynthesis of collagen VII is an extract of Bertholletia.